Enhancement of the Thermostability of Microbacterium Esterase by Combinatorial Rational Design

The esterase EstSIT01 from Microbacterium can catalyze the asymmetric hydrolysis of meso-dimethyl ester to produce the crucial chiral intermediate (4S, 5R)-hemimethyl ester for d-biotin synthesis. Despite its high yields and stereoselectivity, the low thermostability of EstSIT01 limits its practical application. Herein, two kinds of rational strategies were combined to enhance the thermostability of EstSIT01. Based on the Surface Residue Substitution (SRS) method, two variants (G215A and G316A) with improved thermostability and one mutant (G293A) with superior activity were identified from nine candidates. According to the Consensus Mutation method, two mutants (E301P and A332P) with enhanced thermostability were identified from six candidates. However, the combined mutation failed to yield mutants surpassing the best single mutant, E301P, in terms of thermostability. The combined mutant E301P/G215A and E301P/G215A/G293A exhibited a slight enhancement in enzyme activity relative to E301P, while also exhibiting improved thermostability compared to the wild-type EstSIT01. Compared with the wild-type esterase, the thermal inactivation half-lives (t_1/2) of mutant E301P were enhanced 1.4-fold, 2.4-fold and 1.8-fold at 45 °C, 55 °C, and 65 °C, respectively. The optimal reaction temperature and pH for mutant E301P remained consistent with those of the wild type, at 40 °C and 10.0, respectively. The K_m of E301P was 0.22 ± 0.03 mM and the k_cat was 5.1 ± 0.28 s^-1. Further analysis indicated that the free energies of G215A, G293A and E301P were decreased by 0.91, 0.308 and 1.1049 kcal/mol, respectively, compared to the wild-type EstSIT01. The interaction analysis revealed that the substitution of glutamic acid with proline at position 301 enhanced the hydrophobic interactions within the protein. The decreased free energies and the increased hydrophobic interactions were well correlated with the enhanced stability in these mutants.